Fig. 5: Transcriptional profiles of cells recruited in the acute and repeated ARC ensembles during the encoding of cocaine-context associative memories.

a Experimental workflow for CPP ensemble tagging, FANS and snRNAseq in Arc-CreER^T2::Sun1 mice. 4-OHT 4-Hydroxytamoxifen, 1 P 1 Pairing, 5 P 5 Pairings. FANS Fluorescence-activated nuclei sorting. b snRNAseq analysis workflow for pairwise analyses of differentially expressed genes (DEGs). Acu. Acute, Rep. Repeated. c UMAP reduction and cell type annotation of all collected nuclei (n = 11,539). d Proportion of nuclei from each treatment/FANS status combination in each cell type cluster. Exact numbers are available in Supplementary Fig. 8c. χ² tests: Acute: χ² = 284.23, df = 4, p < 0.001. Repeated: χ² = 74.26, df = 4, p < 0.001. FDR-adjusted p-values correspond to standardized Pearson’s residuals. ***p < 0.001 GFP+ vs GFP-, Acute; ^##p < 0.01 GFP+ vs GFP-, Repeated^. e, Expression heatmaps of GFP+ vs. GFP- DEGs in D1-MSNs in acute (top) and repeated (bottom) conditioning groups, along with 3 most significantly enriched IPA canonical pathways (** FDR < 0.01, *** FDR < 0.001). f Expression heatmaps of GFP+ vs. GFP- DEGs in D2-MSNs in acute (top) and repeated (bottom) conditioning groups, along with 3 most significantly enriched IPA canonical pathways (** FDR < 0.01, *** FDR < 0.001). g Overlap of GFP+ vs. GFP- DEGs between D1- and D2-MSNs in either acute (top) or repeated (bottom) conditioning groups (Fisher’s exact test p-values). h Overlap of GFP+ vs. GFP- DEGs between acute and repeated conditioning groups in D1-MSNs (top) and D2-MSNs (bottom) (Fisher’s exact test p-values). i Expression heatmaps of DEGs in GFP+ nuclei between repeated and acute groups in D1-MSNs (top) and D2-MSNs (bottom), along with corresponding 3 most significantly enriched IPA canonical pathways (** FDR < 0.01, *** FDR < 0.001). j Expression dot plots of selected D1-MSN DEGs involved in neuronal physiology and signaling (*FDR < 0.05). k Most significant IPA predicted upstream regulator proteins across comparisons in D1- and D2-MSNs. l Most significant IPA predicted upstream regulator chemical substances across comparisons in D1- and D2-MSNs. Created in BioRender. Parise, E. (2025) https://BioRender.com/voi7l54.